The C. elegans mapping locus rol-9 is encoded by a gain-of-function mutation in mlt-11

We mapped rol-9 to the mlt-11 locus (encoded by the gene W01F3.3) on the far-right end of chromosome V. The canonical allele of rol-9, sc148, is an in-frame deletion in a conserved exon of the protein that creates a gain-of-function roller phenotype. sc148 deletes a short peptide of unknown function conserved in nematodes.

We precisely mapped the mutation causing rol-9(sc148) using whole genome sequencing. There was only one mutation annotated to have high or moderate functional effect on the far-right arm of LGV: a 205 basepair deletion (V:20,669,331-20,669,536, Figure 1B) combined with a four-base insertion (GGGG, Figure 1C). The mutation is found in exon 7 of mlt-11, which is conserved in all splice isoforms, and replaces 68 residues with an arginine and glycine.
The deletion overlaps only 3 amino acids of a Kunitz-type inhibitor domain in the protein, and the deleted sequence matches no conserved domains in the NCBI Conserved Domain Database (Marchler-Bauer et al., 2015). A 13aa peptide in the deletion is highly conserved in the Rhabditina clade ( Figure 1D). This peptide (IRTTQGIRITPNG) is almost perfectly conserved in Caenorhabditis, Haemonchus, and Ancylostoma, but is not conserved in Pristionchus. In all the species studied, this peptide is the only conserved sequence between the second and third Kunitz protease inhibitor domains. It is likely that the peptide was introduced into mlt-11 in the most recent common ancestor of Haemonchus and Caenorhabditis, after the split with Pristionchus.
Most genes with a roller phenotype encode structural constituents of the cuticle or collagens. It isn't clear how sc148, a gain of function mutation in MLT-11, a molting protease inhibitor, induces a Rol phenotype, though it seems likely that the conserved peptide deleted in sc148 may be playing a Rol.

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Fosmid rescue. rol-9(sc148) adult hermaphrodites were injected with either the full-length W01F3 fosmid or restriction fragments in which the fosmid was cut with either SacI, KpnI, or AatII. Fosmids were co-injected with a fluorescent marker to identify lines with extrachromosomal array transmission into the F2 generation. Transmitting lines were scored for rescue. EG1920 contains an extrachromosomal array containing the full-length W01F3 fosmid.
Whole genome sequencing and data analysis. rol-9(sc148) animals were grown on HB101 plates until near starvation, at which point genomic DNA was extracted using a Qiagen DNeasy Blood and Tissue kit. Genomic DNA libraries were prepared using a Nextera transposase kit (Illumina) and were sequenced on an Illumina NovaSeq with 2×150 base reads. Library prep and sequencing were performed by the Huntsman Cancer Institute Genomics Core facility. Alignment and analysis of sequencing reads was performed by an in-house software pipeline. Briefly, reads were aligned to the C. elegans genome (WBcel276) using bwa (Li and Durbin, 2009) and processed with samtools (Li, 2011). Basecalling was performed using GATK4 (McKenna et al., 2010), and homozygous mutations with high genotype quality (GQ>20) were annotated with SNPeff (Cingolani et al., 2012). We identified the deletion in W01F3.3 algorithmically using smoove (Pedersen et al., 2020) and by eye using the Integrative Genome Viewer (Robinson et al., 2011).